The role of local and remote amino acid substitutions for optimizing fluorescence in bacteriophytochromes: A case study on iRFP

David Buhrke, Francisco Vélazquez Escobar, Luisa Sauthof, Svea Wilkening, Nico Herder, Neslihan N. Tavraz, Mario Willoweit, Anke Keidel, Tillmann Utesch, Maria-Andrea Mroginski, Franz-Josef Schmitt, Peter Hildebrandt (+3 others)
Bacteriophytochromes are promising tools for tissue microscopy and imaging due to their fluorescence in the near-infrared region. These applications require optimization of the originally low fluorescence quantum yields via genetic engineering. Factors that favour fluorescence over other non-radiative excited state decay channels are yet poorly understood. In this work we employed resonance Raman and fluorescence spectroscopy to analyse the consequences of multiple amino acid substitutions on
more » ... uorescence of the iRFP713 benchmark protein. Two groups of mutations distinguishing iRFP from its precursor, the PAS-GAF domain of the bacteriophytochrome P2 from Rhodopseudomonas palustris, have qualitatively different effects on the biliverdin cofactor, which exists in a fluorescent (state II) and a non-fluorescent conformer (state I). Substitution of three critical amino acids in the chromophore binding pocket increases the intrinsic fluorescence quantum yield of state II from 1.7 to 5.0% due to slight structural changes of the tetrapyrrole chromophore. Whereas these changes are accompanied by an enrichment of state II from ~40 to ~50%, a major shift to ~88% is achieved by remote amino acid substitutions. Additionally, an increase of the intrinsic fluorescence quantum yield of this conformer by ~34% is achieved. The present results have important implications for future design strategies of biofluorophores.
doi:10.14279/depositonce-8640 fatcat:uxlturvifvcoppshmlkyhbxiuq